Illumina NovaSeq 6000 sequencing; ChIP-seq mapping of gH2AX, PanKcr, and H3K9cr at AsiSI-induced double-strand breaks in control and CDYL1-depleted U2OS-DIvA cells
ChIP-seq mapping of gH2AX, PanKcr, and H3K9cr at AsiSI-induced double-strand breaks in control and CDYL1-depleted U2OS-DIvA cells
U2OS DiVA cells were plated at 15cm and allowed to reach 80% confluency before treatment/collection U2OS-DIvA (AsiSI-ER-U20S) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % heat-inactivated fetal bovine serum (Gibco), 2mM L-glutamine (Gibco), 100unit/mL penicillin, 100μg/mL streptomycin (Gibco) and 1mM Sodium Pyruvate in the presence of 400μg/mL G418 at 37°C and 5% CO2.  Cells were treated with 300nM 4OH for 4 hrs to induce AsiSI-dependent DSBs.  Cells were fixed with 1% PFA for 10 minutes at room temperature, and cross-linking was stopped with 0.125 M Glycine for 5 min. After cell lysis, DNA was sheared to the size of 300–500 bp using a Vibra cell sonicator (15 sec ON, 30 sec OFF, 35% duty, 20 cycles). Five percent of each supernatant was used as input control and processed with the cross-linking reversal step. Chromatin was immunoprecipitated using 2ug of the indicated antibodies and Protein A/G magnetic beads. Following reverse cross-linking; the precipitated DNA was purified using the Macherey-Nagel Nucleospin kit Chip-seq libraries were prepared at the Crown Genomics institute of the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science starting from 1-20ng of DNA as previously described (Blecher-Gonen R et al. Nat. prot. (2013)).  Libraries were quantified by Qubit (Thermo fisher scientific) and TapeStation (Agilent). Libraries average peak size was ~500bp.